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The identification of a protein involved in triggering a cell’s adaptive responses to hypoxia could pave the way to new treatments for diseases caused or exacerbated by reduced oxygen levels, scientists claim. A team led by scientists at the University of California, San Francisco (UCSF), and Gladstone Institute of Neurological Disease has discovered that p75 neurotrophin receptor (p75NTR) undergoes hypoxia-induced cleavage to generate peptides that trigger the stabilization of HIF-1α, a transcription factor known to control a cell’s genetic response to hypoxia.

Working with researchers at the University of Glasgow in the U.K and UCSF’s department of neurology, the Gladstone scientists used an animal model of hypoxia-induced retinopathy to demonstrate that knocking out p75NTR significantly reduces the degree of abnormal neovascularization that otherwise occurs after temporarily reducing oxygen supply to the eye.

Reporting their findings in Molecular Cell, the authors suggest that targeting p75NTR might represent an approach to treating hypoxia-related disorders that operates upstream of HIF-1α. Their paper is titled “Oxygen-Dependent Cleavage of the p75 Neurotrophin Receptor Triggers Stabilization of HIF-1α.”

Aerobic organisms apply sophisticated processes at the cellular level to make sure that adequate oxygen supply is maintained for metabolic functions, and a heterodimeric transcription factor, hypoxia-inducible factor 1α (HIF-1α) represents the master regulator of cellular adaptation to hypoxia in a range of diseases, such as ischemic disorders and cancer, the team explains. HIF-1α responds to hypoxia by switching on the transcription of a range of genes involved in regulating biological processes such as cellular proliferation, survival, angiogenesis, energy, and metabolism.

In conditions of high oxygen levels, HIF-1α itself is degraded by prolyl hydroxylases (PHDs), but when oxygen levels drop, PHDs become inactive due to the upregulation of the seven in abstentia homolog 2 (Siah2) gene, and this results in the instant stabilization of HIF-1α, so it can orchestrate genetic response to hypoxia.

What isn’t yet known, however, the authors continue, is what upstream proteins are required for Siah2-mediated stabilization of HIF-1α. To look at this more closely the team investigated how p75NTR signaling impacts on HIV-1α-mediated hypoxic response. p75NTR is a member of the tumor necrosis factor receptor superfamily, and is upregulated during development as well as in pathologic conditions including CNS injury and disease. Previous research has indicated that p75NTR is widely expressed in non-neuronal tissues and mediates a broad range of cellular functions including including apoptosis, differentiation, myelination, and extracellular matrix remodeling.

The protein undergoes α-secretase and then γ-secretase-dependent intramembrane proteolysis to liberate a soluble intracellular domain (p75ICD) that has signaling functions in processes including apoptosis, neurite outgrowth, and the transcriptional activation of cell-cycle genes. Indeed, the team notes, “combinations of ligand binding, co-receptor interactions, intracellular compartmentalization, and regulated intramembrane proteolysis determine which cytoplasmic partners p75NTR recruits to mediate its pleiotropic cellular functions.”

p75NTR plays a crucial role in liver function, lung fibrosis, and cancer, and given that HIF-1α-mediated hypoxic response is also a hallmark of fibrotic diseases and tumorigenesis, the researchers investigated whether p75NTR might itself act as a regulator of the hypoxic response. Their first step was to compare the protein levels of HIF-1α in p75NTR+/+ (WT) and p75NTR-/- mouse embryonic fibroblasts (MEFs) and cerebellar granule neurons (CGNs)—which normally express p75NTR at equivalent levels—under normoxic and hypoxic conditions.

This indicated that during hypoxia, HIF-1α stabilization was 68% lower in p75NTR-/- MEFs compared with WT cells. Loss of endogenous p75NTR in primary CGNs also decreased HIF-1α stabilization by 55%. Conversely, under normoxic conditions, HIF-1α was constitutively degraded in both WT and p75NTR-/- cells. Supporting a role for p75NTR, the researchers also found that lentiviral delivery of full-length p75NTR (p75FL) to p75NTR cells rescued HIF-1α stabilization. And while stabilized HIF-1α normally translocates to the nucleus during hypoxia, the investigators found that nuclear accumulation of HIF-1α was reduced in p75NTR-/- cells, and the expression of HIF-1α-triggered mRNAs, including GLUT1, PHD3 and VEGF, was reduced by about 50–70%.

Significantly, gene-expression analysis showed no significant differences between WT and p75NTR-/- cells in HIF-1α RNA itself, “indicating that HIF-1α expression is regulated at the protein level in p75NTR-/- cells in response to hypoxia,” the investigators note.

Treating p75NTR-/- cells with a proteosomal inhibitor MG132 increased levels of HIF-1α to those seen in WT cells, which suggests that the defect in HIF-1α stabilization in the p75NTR-/- cells results from excessive proteasomal degradation. This notion was supported by use of desferroxamine (DFO), an iron chelator that inhibits the PHD activity, which restored both the abundance and nuclear accumulation of HIF-1α protein in p75NTR-/- cells to that of WT cells, the team reports. “Thus, increased PHD activity appears to be responsible for the impaired HIF-1α stabilization in p75NTR-/- cells under hypoxia."

Confirmation that P75NTR interacts directly with Siah2 was demonstrated using co-immunoprecipitation in whole brain extracts, MEFs, and N2a neuroblastoma cells in hypoxic conditions, while deletion mapping mutagenesis and co-immunoprecipitation studies indicated that the interaction of p75NTR with Siah2 appears to be oxygen dependent, but independent of ligand binding.

To identify which p75NTR domains are involved in Siah2 interaction, the team carried out mapping studies with deletion mutants. These results indicated that Siah2 interacts with the juxtamembrane region of p75NTR (between residues 275 and 343). Subsequent studies using P75NTR mutants showed that a construct lacking the juxtamembrane domain (p75Δ151) failed to stabilize HIF-1α upon hypoxia. Binding studies using peptide arrays comprising overlapping peptides spanning the entire sequence of Siah2 in addition indicated that the juxtamembrane region of p75NTR actually interacts with the evolutionarily conserved RING and zinc finger domains of Siah2.

Because Siah2 is upregulated in hypoxia and induces a positive feed-forward mechanism to enhance HIF-1α stability, and given that steady-state levels of Siah2 are regulated by its RING domain, which controls Siah2 autoubiquitination and thus its self-degradation, the team hypothesized that p75NTR increases Siah2 abundance in hypoxia by regulating its autoubiquitination. This was borne out by the finding that in p75NTR-/- cells Siah2 was reduced by over 50%, even though p75NTR-/- and WT cells demonstrated no differences in the transcriptional regulation of Siah2 during hypoxia. Transfection of p75NTR-/- cells with a Siah2 expression vector, however, rescued the deficit in HIF-1α accumulation, adding weight to the notion that p75NTR regulates HIF-1α stabilization through Siah2, the investigators state.

These results, combined with the observations that under hypoxic (but not normoxic) conditions p75FL dramatically reduced the in vivo autoubiquitination of Siah2, and that inhibition of proteasomal activity with MG132 increased the accumulation of ubiquitinated forms of Siah2, suggested that “in low-oxygen levels, p75NTR regulates Siah2 turnover by decreasing Siah2 self-degradation.”

The researchers next turned their attentions to investigating how oxygen levels regulate p75NTR. They found that while hypoxia didn’t increase p75NTR RNA or protein levels, it did stimulate the intramembrane proteolysis of p75NTR. In fact, hypoxia induced the formation of p75ICD in MEFs transfected with p75FL within five hours, and hypoxia-induced intramembrane cleaves were also observed for endogenous p75NTR in WT MEFs and in N2a neuroblastoma cells. Treating cells with synthetic inhibitors of either α-secretase or γ-secretase abolished the formation of the p75NTR proteolytic fragments, “suggesting that metalloprotease-mediated shedding precedes ICD release during hypoxia,” they remark. Furthermore, studies with a p75NTR mutant that is resistant to γ-secretase showed that hypoxia-induced intramembrane cleavage of p75NTR is necessary for HIF-1α stabilization and Siah2 accumulation.

A set of experiments was then carried out to evaluate the biological significance of p75NTR in terms of HIF-1α stabilization and hypoxic response in vivo, using an established oxygen-induced retinopathy (OIR) model of retinal hypoxia. In OIR, hyperoxia produces regression of the vascular network (vaso-obliteration) in the center of the retina, which becomes hypoxic upon return to normoxia. This vaso-obliteration results in compensatory neovascularization characterized by chaotically orientated and inefficient blood vessels that lead to pathological angiogenesis, the researcher explain.

Both WT and p75NTR-/- mice were subjected to hyperoxia from postnatal day seven (p7) to P12, followed by a return to ambient air (normoxia) for another five days. Analysis of the animals showed that in the p75NTR-/- mice, the vaso-obliterated area was 45% smaller than in WT mice, and pathological neovascularisation was reduced by 43%.

Then, because p75NTR-/- mice are known to show deficits in vaso-obliteration and angiogenesis similar to mice with HIF-1α depletion, they looked at whether p75NTR regulates HIF-1α stabilization in vivo. They found elevated levels of HIF-1α in the ganglion cell layer (GCL) after hypoxia, and also increased levels of the HIF-1α target gene VEGF, which is implicated in ischemic retinopathies by promoting pathological neovascularisation.

Further analysis of p75NTR cleavage in the retina showed that p75NTR proteolytic fragments formed as early as six hours after retinal hypoxia, and their formation was sustained five days after hypoxia. “In accordance with our in vitro studies, hypoxia did not alter p75NTR protein levels in vivo, suggesting that hypoxia primarily regulates p75NTR by inducing its proteolytic cleavage,” they add. Critically, p75NTR-/- mice exhibited a dramatic reduction of HIF-1α stabilization in the retina after hypoxia compared to WT controls, and while VEGF was upregulated in the retina of WT mice after hypoxia, its expression didn’t change in the p75NTR-/- animals.

Finally, the researchers investigated whether in vivo inhibition of p75NTR cleavage with α-secretase or γ-secretase inhibitors had an impact on the HIF-1α-mediated hypoxic response. As expected, they found that administration of either type of inhibitor decreased HIF-1α stabilization and VEGF induction in the retina, and prevented the formation of proteolytic fragments, including p75ICD.

“Our findings reveal a previously unrecognized mechanism for the regulation of the hypoxic response initiated by oxygen-dependent cleavage of p75NTR, which triggers stabilization of HIF-1α through the ubiquitin ligase Siah2,” the authors conclude. “These results demonstrate a crucial role for the oxygen-dependent intramembrane cleavage of p75NTR in the regulation of HIF-1α-mediated angiogenesis in vivo...Since the HIF pathway has been proposed as a potential target for therapeutic intervention in ischemic retinopathies, p75NTR might be a therapeutic target upstream of HIF-1α.” Moreover, they add, while their in vivo studies were focused on retinal pathology, p75NTR expression is not restricted to the CNS, so it’s possible that p75NTR might contribute to the hypoxic response in other tissues. “Regulated intramembrane cleavage of p75NTR and subsequent interaction with Siah2 might be targets for therapeutic intervention to modulate the hypoxic response in ischemia and cancer characterized by dysregulation of HIF-1α."

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